Anti-JNK2抗体[EP1595Y] (ab76125)

Anti-JNK2 antibody [EP1595Y] 图像

• 

  Immunocytochemistry/ Immunofluorescence - Anti-JNK2 antibody [EP1595Y] (ab76125)

  Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling JNK2 with purified ab76125 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

  Control: PBS only

• 

  Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125)
  
  
  Predicted band size : 48 kDa
  

  Lane 1: Wild-type HAP1 cell lysate (20 µg)
  Lane 2: JNK2 knockout HAP1 cell lysate (20 µg)
  Lane 3: HeLa cell lysate (20 µg)
  Lane 4: MCF7 cell lysate (20 µg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab76125 observed at 54 kDa. Red - loading control, ab8245, observed at 37 kDa.
  
  ab76125 was shown to specifically react with JNK2 when JNK2 knockout samples were used. Wild-type and JNK2 knockout samples were subjected to SDS-PAGE. ab76125 and ab8245 (loading control to GAPDH) were diluted 1/2500 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

• 

  Immunoprecipitation - Anti-JNK2 antibody [EP1595Y] (ab76125)

  JNK2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit monoclonal to JNK2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

  The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

  Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab76125.

  Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

  Band: 48kDa; JNK2

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - JNK2 antibody [EP1595Y] (ab76125)
  ab76125 at 1/100 dilution staining JNK2 in human breast carcinoma by Immunohistochemistry, Paraffin-embedded tissue.
• 

  Western blot - JNK2 antibody [EP1595Y] (ab76125)
  Anti-JNK2 antibody [EP1595Y] (ab76125) at 1/50000 dilution + HeLa cell lysate at 10 µg
  
  Secondary
  goat anti-rabbit-HRP at 1/1000 dilution
  Developed using the ECL technique
  
  Predicted band size : 48 kDa
  Observed band size : 54 kDa (why is the actual band size different from the predicted?)
  Additional bands at : 46 kDa (possible isoform).
• 

  Immunocytochemistry/ Immunofluorescence - Anti-JNK2 antibody [EP1595Y] (ab76125)
  ICC/IF image of ab76125 stained HeLa cells. The cells were 4% formaldehyde (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76125, neat) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  
  
• 

  Flow Cytometry-Anti-JNK2 antibody [EP1595Y](ab76125)
  Overlay histogram showing HeLa cells stained with ab76125 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76125, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.